The major objectives are to determine mechanisms of replicative (aim 1) and transcriptional (aim 2) mutagenesis relevant to the development of human disease (such as cancer) and to also determine the relationship between dysregulation of DNA repair pathways (i.e. base excision repair) and tumor development (aim 3). Aim 1 accomplishments. Redox stress is a major hallmark of cancer. Analysis of thousands of sequenced cancer exomes and whole genomes revealed distinct mutational signatures which can be attributed to specific sources of DNA lesions. Clustered mutations discovered in several cancer genomes reflect the persistence of single strand (ss)DNA intermediates arising as a result of various processes of DNA metabolism. Only one clustered mutation signature caused by a subclass of ssDNA-specific APOBEC cytidine deaminases was clearly defined so far. Others remain to be elucidated. We report here deciphering of the mutational spectra and mutational signature of redox stress in ssDNA of budding yeast and the signature of aging in human mitochondrial DNA. We found that the prevalence of C to T substitutions is a common feature of both signatures. Measurements of the frequencies of hydrogen peroxide-induced mutations in proofreading-defective yeast mutants allowed to conclude that hydrogen peroxide-induced mutagenesis is not the result of increased DNA polymerases misincorporation errors but rather is caused by direct damage to DNA. Proteins involved in modulation of chromatin status appeared to play a significant role in prevention of redox stress-induced mutagenesis, possibly by facilitating protection by modification of chromatin structure. These findings for the first time allow to enable the search for the mutational signature of redox stress in cancers and in other pathological conditions and could potentially be used for informing therapeutic decisions. In addition, such mutational signatures could be imprinted in evolutionary history of species and may further advance our understanding of mechanisms that drive evolution. Aim 2 accomplishments. A system is being established to determine whether transcriptional mutagenesis is responsible for driving the acquisition of antibiotic resistance in pathogenic microorganisms. All cells, regardless of their proliferation state, constantly experience DNA damage from environmental and endogenous sources. DNA base damages can often have miscoding properties and be mutagenically bypassed by DNA polymerase. The well-established mutagenic potential of DNA damage during DNA replication has been linked to genomic instability and a variety of diseases. However, largely quiescent cells are also at risk from the potentially deleterious consequences of DNA damage, as many of these lesions can also be bypassed by RNA polymerase, giving rise to mutant RNA molecules. This process is termed transcriptional mutagenesis (TM). We have shown that many types of common, environmentally relevant DNA damages, especially oxidative base lesions, cause TM via RNA polymerase miscoding and, if such events occur on the template strands of transcribed genes, can result in protein sequence changes that can alter the phenotypes of non-dividing bacterial cells. In mammalian cells TM can cause oncogene activation and subsequent stimulation of growth-positive signal transduction pathways. Since analogous, incorrect bases are often incorporated opposite the lesion by both DNA and RNA polymerases, if a phenotype conferred by the transcriptional mutation results in DNA replication, one of the resulting daughter cells could acquire a permanent DNA mutation, and thus permanent establishment of the phenotype. This mechanism, which can potentially allow quiescent cells to enter the cell cycle and acquire novel mutations, has been termed retromutagenesis (RM). TM and RM may have a deleterious impact on human health by contributing to the etiology of diseases such ascancer, neurodegenerative and developmental disorders as well as giving rise to antibiotic-resistant pathogenic bacteria. Our group previously demonstrated that TM-driven RM can result in escape from growth selection (reversion to prototrophy) in the Gram negative bacterium Escherichia coli. RM is likely to be a universal method of evolutionary adaptation, which enables the emergence of new mutants from mutations acquired during counter-selection rather than beforehand, and it may have a major role in the development of antibiotic resistance in microorganisms. Pseudomonas aeruginosa (Pa) represents an ideal bacterium to test the hypotheses that RM plays an important role in the acquisition of antibiotic resistance as it is an important human pathogen and new approaches are needed to understand how antibiotic resistance emerges. The process of TM may be particularly important during chronic infection when the bacteria encounter known mutagens such as ROS and other human neutrophil components that have mutagenic properties such as the human cathelicidin LL-37. To address this question, we have modified the systems we developed for E. coli to test the emergence of ciprofloxacin (fluoroquinolone antibiotics (FQ)) resistance in Pa. In addition, the results from this project will also reveal the feasibility of employing RNA polymerase inhibitors such as rifampicin in combination with FQ antibiotics to reduce or eliminate the emergence of resistant strains. Thus, this project has significant translational potential. We anticipate completion of the RM detection system and determination of the contribution of RM to FQ resistance during the next fiscalyear. Aim 3 accomplishments (note: these studies were partly conducted at the NIEHS and partly conducted at Emory University (where Paul Doetsch was a faculty member prior to recruitment to the NIH). Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We also obtained evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer. We have recently generated data showing that overexpression of NTHL1 sensitizes cells to cisplatin and the PAR inhibitor olaparib.